sFasL Assay Kit

N° du produit ABIN955773

Détail du produit

Antigène: sFas Ligand (sFasL) (Soluble Factor-Related Apoptosis Ligand (sFasL))

Méthode de détection: Colorimetric

Type d'échantillon: Cell Culture Supernatant, Plasma, Serum

Specificité: The interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into a serum sample. There was no detectable cross-reactivity.

Sensibilité: The limit of detection of sFas Ligand defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus two standard deviations) was determined to be 0.07 ng/mL (mean of 6 independent assays).

Ingrédients: 1 aluminum pouch with a Microwell Plate coated with Monoclonal Antibody (mouse) to human sFas Ligand.
1 vial (100 µL) Biotin-Conjugate anti-sFas Ligand monoclonal antibody (mouse)
2 vials, sFas Ligand Calibrator, lyophilized, 20 ng/mL upon reconstitution
1 vial (150 µL) Streptavidin-HRP
1 bottle (50 mL) Wash Buffer Concentrate 20X (PBS with 1% Tween 20)
1 vial (5 mL) Assay Buffer Concentrate 20X (PBS with 1% Tween 20)
1 vial (12 mL) Sample Diluent (buffered protein matrix)
1 vial (15 mL) Substrate Solution
1 vial (12 mL) Stop Solution (1M Phosphoric acid)
1 vial (0.4 mL each) Blue Dye, Green Dye, Red Dye
4 adhesive Plate Seals.

Matériel non inclus: 5 mL and 10 mL graduated pipettes
10 µL to 1,000 µL adjustable single-channel micropipettes with disposable tips
50 µL to 300 µL adjustable multi-channel micropipette with disposable tips
Multi-channel micropipette reservoir
Beakers, flasks, cylinders necessary for preparation of reagents
Device for delivery of Wash Solution (multi-channel wash bottle or automatic wash system)
Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wavelength)
Glass-distilled or de-ionized water
Statistical calculator with program to perform linear regression analysis.

Information d'application

Plaque: Pre-coated

Préparation des réactifs:

Prepare Wash Buffer and Assay Buffer before starting with the test procedure.

A. Wash Buffer: If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. Pour entire contents (50 mL) of the Wash Buffer Concentrate into a clean 1,000 mL graduated cylinder. Bring final volume to 1,000 mL with glass-distilled or de-ionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at 4° to 25°C. Please note that the Wash Buffer is stable for 30 days.

B. Assay Buffer: Mix the contents of the bottle well. Add contents of Assay Buffer Concentrate (5.0 mL) to 95 mL distilled or de-ionized water and mix gently to avoid foaming. Store at 4°C. Please note that the Assay Buffer is stable for 30 days.

C. Preparation of Calibrator: Dissolve lyophilized Calibrator by addition of distilled water. Reconstitution volume is stated on the label of the vial. Swirl gently to ensure complete solubilization.

D. Preparation of Biotin-Conjugate: Dilute the Biotin-Conjugate 1:100 just prior to use with diluted Assay Buffer in a clean plastic tube. Mix the contents of the tube well. Please note that the Biotin-Conjugate should be used within 30 minutes after dilution.

E. Preparation of Streptavidin-HRP: Make a 1:200 dilution of the concentrated Streptavidin-HRP solution with diluted Assay Buffer as needed.

F. Addition of color-giving reagents: Blue Dye, Green Dye, Red Dye In order to help our customers to avoid any mistakes in pipetting the sFas Ligand ELISA, we now offer a new tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colors to each step of the ELISA procedure. This procedure is optional, does not in any way interfere with the test results, and is designed to help the customer with the performance of the test, but can also be omitted, just following the package insert.

Préparation de l'échantillon:

Cell culture supernatants, human serum, EDTA, or heparinized plasma, amniotic fluid, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples must be stored frozen at -20°C to avoid loss of bioactive sFas Ligand. If samples are to be run within 24 hours, they may be stored at 4°C. Avoid repeated freeze-thaw cycles. Prior to assay, frozen sera or plasma should be brought to room temperature slowly and mixed gently and properly diluted with Sample Diluent.

Calcul des résultats:

Calculate the average absorbance values for each set of duplicate calibrators and samples. Duplicates should be within 20 percent of the mean.
Create a calibration curve by plotting the mean absorbance for each calibrator concentration on the ordinate against the sFas Ligand concentration on the abscissa. Draw a best-fit curve through the points of the graph.
To determine the concentration of circulating sFas Ligand for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the calibration curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding sFas Ligand concentration.
For samples that have been diluted 1:2 according to the instructions given in this manual, the concentration has to be multiplied by the dilution factor (x2).
It is suggested that each testing facility establishes a control sample of known sFas Ligand concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Since exact conditions may vary from assay to assay, a calibration curve must be established for every run.
Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.
Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use.
Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Wash Buffer, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods.
The use of immunotherapy has significantly increased the number of patients with human anti-mouse IgG antibody (HAMA). HAMA may interfere with assays utilizing mouse monoclonal antibodies leading to both false positive and false negative results. Serum samples containing antibodies to mouse immunoglobulins can still be analyzed in such assays when mouse immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) are added to the Sample Diluent.

Stock: 4 °C

Stockage commentaire: Store kit reagents at 4°C. Immediately after use remaining reagents should be returned to cold storage (4°C). Expiration date of the kit and reagents is stated on labels. The expiration date of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling. Aliquots of serum samples (unspiked or spiked) were stored at -20°C and thawed up to 5 times, and sFas Ligand levels determined. There was no significant loss of sFas Ligand by freezing and thawing up to 5 times. serum sample (Aliquots of a spiked or unspiked) were stored at -20°C, 4°C, room temperature (RT) and at 37°C, and the sFas Ligand level determined after 24 hr. There was a loss of sFas Ligand immunoreactivity during storage at 37°C. Therefore higher temperatures during handling the serum samples should be avoided.

Date de péremption: The expiry date is stated on the label.

Détail du antigène

Antigène: sFas Ligand (sFasL) (Soluble Factor-Related Apoptosis Ligand (sFasL))

Autre désignation: sFasL

Sujet: Fas (APO-1, CD95) is a type I membrane protein that belongs to the TNF/nerve growth factor receptor family. Fas mediates apoptosis, the programmed cell death, when it is cross-linked with specific binding partners. The natural binding partner of Fas is its ligand, FasL, which is a 37 kDa type II-membrane protein that belongs to the TNF family which includes TNF, lymphotoxin, TNF-related apoptosis-inducing ligand (TRAIL), CD40 ligand, CD27 ligand, CD30 ligand, and OX40 ligand. FasL is predominantly expressed on activated T-cells and NK cells, thus FasL-mediated cell death is involved in the T or NK cell-mediated cytotoxicity, some pathologic tissue damages, and the regulation of lymphocyte homeostasis. FasL is also expressed in the testis, eye, and some malignant tumor cells, which has been proposed to contribute to their immune-privileged status. A soluble form of FasL (sFasL) is naturally produced by metalloproteinase-mediated processing. The soluble form resulting from this cleavage was shown to induce apoptosis in susceptible cells. Several lines of evidence suggest that sFasL may be involved in the pathogenesis of tissue injury. Circulating sFasL is elevated in the serum of patients with various diseases. Markedly elevated levels of sFasL have been shown in TEN (Toxic Epidermal Necrolysis, Lyell's Syndrome) patients' sera. FasL furthermore turned out to be a sensor for DNA damage in skin cancer. Elevated expression levels of FasL have been measured in various proliferative disorders and cancers like esophageal carcinomas, metastasizing colorectal tumors, hepatocellular carcinoma, multiple myeloma, sarcoma, Non-Hodgkin's lymphoma and nasal lymphoma. Liver dysfunction was shown to be paralleled by increased sFasL levels as well as kidney damage. FasL is discussed to be involved in the pathogenesis of autoimmune diseases, especially the concentrations of sFasL are remarkably higher in the sera and synovial fluids of patients with severe rheumatoid arthritis as compared to normal controls. Increased levels of soluble FasL in the serum of graft-versus-host-disease patients make it a good marker for treatment of the disease. Levels of soluble Fas Ligand in bronchoalveolar lavage (BAL) fluid of humans with acute lung injury (ARDS) and serum levels in congestive heart failure and multiple organ failure were significantly higher than in healthy controls. Cerebrospinal fluid from patients with severe brain injury contains high concentrations of FasL. Elevations of serum FasL levels in hematological disorders and HIV infections are furthermore described.An anti-sFas Ligand monoclonal coating antibody is adsorbed onto microwells. sFas Ligand present in the sample or calibrators binds to antibodies adsorbed to the microwells, a biotin-conjugated monoclonal anti-sFas Ligand antibody is added and binds to sFas Ligand captured by the first antibody. Following incubation, unbound biotin-conjugated anti-sFas Ligand is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti-sFas Ligand. Following incubation, unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of sFas Ligand present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A calibration curve is prepared from seven sFas Ligand calibrator dilutions and sFas Ligand sample concentration determined.